Naturally Selected Anti-CD7 CAR-T Cells without Additional Genetic Manipulations As a Potentially Superior Therapy for T-Cell Malignancies

• Published in: Blood 2021-11, Vol.138 (Supplement 1), p.1696-1696
• Main Authors: Liu, Ying, Li, Weijing, Wang, Lin, Ba, Min, Wang, Qinglong, Lu, Peihua, Liu, Lihong, Li, Jianqiang
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2021-149384

Introduction To prevent CAR-T fratricide, anti-CD7 CAR (7CAR) T cells used for treating T-cell malignancies are often modified by CD7 ablation via CRISPR/CAS9 gene editing or by co-expression of a CD7-specific protein expression blocker. Both methods require additional genetic manipulations of CAR-T. Here we transduce 7CAR into bulk T cells without CD7 disruption and thereafter allow CAR-T cells to emerge in vitro after fratricidal “natural selection”. The biological characteristics of these naturally selected anti-CD7 CAR (NS7CAR) T cells and their potential advantages in treating patients with T-cell malignancies are described. Methods The percentage of CD3 +CD7 - T cells in peripheral blood from either healthy donors (HDs) or patients (PTs) were determined by flow cytometry. Peripheral bulk T cells were positively selected using CD3 magnetic beads, and peripheral CD7 - T cells were negatively selected using CD7 magnetic beads. To avoid contamination from malignant T cells, patients only with CD3 -CD7 + T cell blasts were included in this study. The 7CAR gene cassette comprising of the cDNA of a CD7-specific antibody sequence fused to the coding sequences for the CD8TM-41BB-CD3z signal domains, and the T2A-linked tEGFR was cloned into a lentiviral vector backbone under the control of an EF1α promoter. 7CAR lentiviral transduction of bulk T cells (NS7CAR) or CD7 - T cells (Neg7CAR) were performed two days after CD3/CD28 dynabeads activation. CD7-ablated 7CAR T cells (KO7CAR) were derived by electroporation of bulk T cells with CD7-targeting Cas9-gRNA RNP 24 hours before 7CAR transduction. CAR-T cells were routinely kept in culture for 12 days. The levels of CD7 mRNA, protein, and surface expression were determined respectively by qualitative/quantitative reverse transcription PCR, Western blotting, and flow cytometry. Iv vitro cytotoxic activity for CD7 + tumor cell lines was tested using a flow-cytometry-based cytotoxicity assay. NSG mice engrafted with CCRF-CEM-luciferase cells were used as an animal model to validate the activities of CD7 CAR. Results Three approaches for generating anti-CD7 CAR-T cells were compared: NS7CAR (fratricidal natural selection from bulk T cells after 7CAR transduction), Neg7CAR (7CAR transduction of purified CD7-negative T cells) and KO7CAR (Cas9 RNP CD7 gene ablation). While CD7 - T cells were detectable in HDs of all ages (9.48±0.96%, n=13), we observed a significant increase of this cell population in T-cell acute lympho